Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet: Conventional αβ T Cells in Fetal Skin Largely Demonstrate a Naive, Proliferative Phenotype Live, CD4 + , and CD8 + single-positive cells from 23 weeks fetal skin and adult skin processed for CyTOF were identified by surface markers and analyzed as follows. (A–D) UMAP plots of combined fetal and adult (A) CD4 + and (B) CD8 + T cells colored by skin age. Principal component analysis (PCA) plots demonstrating the distribution of all (C) CD4 + and (D) CD8 + T cells from each individual sample by age. (E) UMAP plots of CD4 + T cells from fetal and adult skin, combined. Cells are labeled and colored by cluster, with clusters A and B constituting conventional CD4 + T cells and cluster C representing Tregs. (F and G) Analogous UMAP plots containing only (F) fetal or (G) adult CD4 + T cells. (H) Heatmap demonstrating relative expression of key markers by CD4 + clusters A, B, and C. (I–K) combined and separated UMAP plots of fetal and adult skin CD8 + T cells, colored by cluster. (L) Heatmap demonstrating relative expression of key markers by each CD8 + cluster. (M–R) Median expression intensity (m.e.i.) of Ki-67 (M and P), CD45RO (N and Q), and CD25 (O and R) for fetal versus adult CD4 + Tconv and CD8 + T cells as revealed by mass cytometric analyses. Each point in (C), (D), and (M)–(R) represents data from an individual donor. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Labeling, Expressing

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet: Subsets of Fetal Skin CD4 + Tconv and CD8 + T Cells Display a Memory Phenotype and Demonstrate Capacity for IFNγ Production Cells were isolated from second trimester fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry. (A) Representative plots demonstrating CD45RO expression by fetal CD8 + T cells (gated on live CD3 + CD8 + CD4 neg ) and CD4 + Tconv (gated on live CD3 + CD4 + CD8 neg Foxp3 neg CD25 lo ). (B and C) Percentage of CD45RO + (B) CD8 + T cells and (C) CD4 + Tconv in fetal versus adult skin. (D–F) Representative histogram (D) and quantification (E and F) of Nur77 MFI on fetal skin CD45RO + versus CD45RA + CD4 + Tconv and CD8 + cells. (G–J) Percentage of CD4 + Tconv producing (G) IL-17A, (H) IL-13, and (I) IFNγ after PMA/ionomycin re-stimulation, and (J) percentage of IFNγ-producing CD8 + T cells. Each point in (B)–(G) represents data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet: Tregs in Human Fetal Skin Demonstrate an Effector Memory Phenotype Twenty-three weeks g.a. fetal torso skin and healthy adult torso skin samples were analyzed for 22 markers using mass cytometry. (A) PCA plot of Tregs (cluster C, Figure 2 ) and CD4 + Tconv (clusters A & B, Figure 2 ) from 23 week fetal skin versus adult skin based on relative expression of key markers as assessed by mass cytometry. (B–E) Graphs showing median expression intensity (m.e.i.) of (B) Foxp3, (C) CD25, (D) PD-1, and (E) CTLA4 by mass cytometry on fetal versus adult skin Tregs. (F and G) Cells were isolated from 17–23 weeks g.a. fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry. (F) Percentage of CD45RO + Tregs in skin by age and (G) accompanying representative flow plots. (H) Representative histogram and (i) quantification of Nur77 MFI on fetal skin CD45RO + versus CD45RA + Tregs. (J) Nur77 in fetal skin CD45RA + CD8 + , CD4 + Tconv, and Tregs. Each point in (A)–(E) represents data from an individual donor. Points in (F), (I), and (J) point represent data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Mass Cytometry, Expressing, Isolation, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature

doi: 10.1016/j.xcrm.2020.100132

Figure Lengend Snippet:

Article Snippet: Anti-human CD25-PECy7 (clone: M-A251) , BD Biosciences , Cat# 557741; RRID: AB_396847.

Techniques: Flow Cytometry, Mass Cytometry, Immunohistochemistry, Recombinant, Staining, Cell Stimulation, Software

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of DCs in vCP172-infected cultures. (A) Immature human DCs were infected with vCP172 (MOI of 10). Infected (+) and uninfected (−) DCs were cultured for 4 days. After culture, the cells were harvested and monitored for the expression of CD25-, CD83-, and CD86-PE (log PE y axes) versus HLA-DR–FITC (x axes). The percentage of large cells expressing CD25 and CD83 above the isotype control are indicated. (B) Immature rhesus macaque DCs were infected with vCP172 (+) (MOI of 10) or not (−) and cultured for 1 to 3 days before being harvested and analyzed. FACS analysis was performed on cells stained with FITC–anti-HLA-DR versus PE-immunoglobulin, -anti-CD25, -CD80, -CD83, or -CD86. Similar data were obtained from more than five experiments with human DCs and three different monkey donors.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Cell Culture, Expressing, Staining

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Induction of maturation is stimulated by viable canarypox virus. (A) Immature human DCs were infected with an MOI of 10 of either vCP180 or the parental strain (ALVAC) or left uninfected (medium). CD25 surface expression by large HLA-DR-positive cells was assessed 4 days after infection (CD25-PE, y axes; HLA-DR–FITC, x axes). The percentage of CD25-positive cells (above isotype control) are indicated in each panel (highlighted by arrowheads). (B) Immature human DCs that had been infected with vCP180 3 to 4 days earlier were sorted into CD25-negative (CD25 neg.) and CD25-positive (CD25 pos.) fractions. Each fraction was then immunostained for intracellular expression of p27 and analyzed by FACS. The percentage of SIV p27-positive cells, above the immunoglobulin control, is shown for each subset. (C) Immature DCs (human) were infected with live (ALVAC) or heat-inactivated (H.I.) ALVAC or left untreated (medium). After 4 days the DCs were examined for CD25 expression by FACS. The percentages of CD25-positive large cells (compared to the isotype control) are shown in each panel. The CD25-positive subset is highlighted by an arrowhead.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Inhibition of maturation of ALVAC-infected DCs by the addition of a caspase 3 inhibitor. A total of 250 or 500 μM Z-DEVD-FMK, or the equivalent dilution of DMSO diluent for the high dose (DMSO), were added to immature DCs 30 min before infection with ALVAC. Inhibition of maturation was assessed by the lack of CD25 expression 4 days after infection using the DMSO-treated cells as the 100% matured population. The data represent the mean percentages of CD25-expression (% CD25 pos.) of three experiments.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Inhibition, Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of uninfected DCs by ALVAC-infected immature DCs. Immature DCs were infected with ALVAC, and free virus was washed out. Uninfected immature DCs that had been stained with the green fluorescent dye CMFDA were added to the infected (unstained) DCs at a ratio of 1:1 (Inf. DCs). As controls, the supernatant from infected cells (collected directly after washing off the virus) was added to CMFDA-stained cells (Sup't) or the green-uninfected cells were kept in medium (Medium). Maturation (highlighted by arrowheads) was assessed by CD25 expression 4 days later. The log PE is expressed on the y axes (CD25 versus the IgG control), and the CMFDA fluorescence intensity is expressed on the x axes. The results from one of two similar experiments are provided.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Staining, Expressing, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses

doi: 10.1186/s13287-015-0050-0

Figure Lengend Snippet: Multiple allogeneic mesenchymal stem cell (MSC) injections result in changes in splenic regulatory T cell percentages. (A-D) There were no significant changes in splenic CD21 + B-cell (A) , CD4 + T-cell (B) , or CD8 + T-cell percentages (C) or CD4/CD8 ratios (D) following multiple MSC injections. (E) There were no significant changes in activated (CD25 + ) lymphocyte proportions. (F) There were significantly higher percentages of splenic FoxP3 + regulatory T cells in the horses injected with bone marrow (BM)-derived MSCs compared with horses injected with adipose tissue (AT)-derived MSCs. Data are presented as mean ± standard error of the mean. * P <0.05.

Article Snippet: The following antibodies were used: mouse-anti-equine CD3 (clone UC F6G 1:250; Jeffery Stott, University of California, Davis, CA, USA) [ ], mouse-anti-human CD21 (clone B-ly4 1:20; BD Pharmingen, San Jose, CA, USA) [ , ], polyclonal goat-anti-human CD25 (clone AF-223; R&D Systems) [ ], rat-anti-mouse/human FoxP3 (clone PCH101; ebioscience, San Diego, CA, USA) [ ], mouse-anti-CD4 (clone HB61A 1:133; VMRD, Pullman, WA, USA) [ ], mouse-anti-equine CD8 (clone F18P 1:500; J. Stott) [ ], and a donkey-anti-mouse secondary when necessary (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).

Techniques: Injection, Derivative Assay

Journal: Advances in Hematology

Article Title: In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33 + Acute Myeloid Leukemia

doi: 10.1155/2012/683065

Figure Lengend Snippet: EBV-CTLs could be stably transduced with the anti-CD33.CAR without alteration in their native immunophenotype and expansion rate. (a) The expression of the anti-CD33.CAR on the surface of EBV-CTLs was evaluated by flow cytometry with a Cy5-conjugated-mAb specific for the CH2CH3 domain of the CAR after 7 days of culture. A representative plot of EBV-CTLs transduction after 7 days of culture is shown. (b) The expression of CD4, CD8, CD3 along with CD56, CD4, and CD8 along with CD25, CD4, and CD8 along with CD45RA, CD4, and CD8 along with CD62L on the surface of EBV-CTLs was evaluated after 30 days of culture by flow cytometry. (c) Proliferation of anti-CD33.CAR-transduced EBV-CTLs compared to unmanipulated EBV-CTLs was evaluated by cell count with Trypan blue exclusion after weekly stimulations at 4 : 1 ratio with either irradiated autologous LCLs. Cells were cultured with low-dose rhIL-2 (20 U/mL). Data shown are mean ± SD of 6 separate experiments.

Article Snippet: The following monoclonal antibodies were used: fluorescein isothiocyanate (FITC)—labeled mAb specific for human CD3, CD4, CD45RA; phycoerythrin (PE)—labeled mAb specific for human CD25, CD28, CD45RO, CD56, CD62L, CD69, CCR7, and TCR γ / δ ; peridin chlorophyll protein (PerCP)—labeled mAb specific for the human CD8 (Becton Dickinson (BD) Biosciences (San Jose, CA)).

Techniques: Stable Transfection, Transduction, Expressing, Flow Cytometry, Cell Counting, Irradiation, Cell Culture

Journal: iScience

Article Title: Defects in NK cell immunity of pediatric cancer patients revealed by deep immune profiling

doi: 10.1016/j.isci.2024.110837

Figure Lengend Snippet:

Article Snippet: Anti-CD25 (clone 2A3, conjugated to 149Sm) , Standard BioTools , Cat#3149010B.

Techniques: Purification, Clinical Proteomics, Recombinant, Blocking Assay, Staining, Saline, Mass Cytometry, Software, Cytometry

Journal: Clinical & Translational Immunology

Article Title: Anti‐C1q autoantibodies from systemic lupus erythematosus patients enhance CD40–CD154‐mediated inflammation in peripheral blood mononuclear cells in vitro

doi: 10.1002/cti2.1408

Figure Lengend Snippet: T cell proliferation, activation and IL‐10 secretion in activated T cells are not affected by bound C1q, bound C1q/anti‐C1q and soluble C1q, respectively, whereas TNF secretion is decreased in the presence of soluble C1q. T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and cultured on bound HSA, bound C1q, bound C1q preincubated with anti‐C1q‐positive systemic lupus erythematosus serum (bound C1q/anti‐C1q), or together with soluble C1q without coating. T cells were activated by tetrameric anti‐CD3/CD28. Cytokines (a) TNF and (b) IL‐10 as well as activation markers (c) CD25 and (d) CD69 were analysed after 24 h by ELISA and flow cytometry, respectively. For proliferation assessment, cells were stained with CFSE prior to the experiment and (e) per cent dividing cells and (f) proliferation index were analysed by flow cytometry after 96 h. Data points represent six different healthy donors used to obtain PBMCs analysed in independent experiments with connecting lines linking data points of a single individual. Median values are shown as solid horizontal lines. The Friedman test with Dunn's posttest correction (all vs bound HSA), * P < 0.05. (c, d) Relative intensity is calculated by normalising MFI of bound C1q, bound C1q/anti‐C1q and soluble C1q to bound HSA. The horizontal dashed line marks the relative change in intensity of 1.0 (Supplementary figure a depicts the gating strategy).

Article Snippet: Staining for surface marker expression was performed for 30 min at 4°C and included the following antibodies: mouse anti‐human CD25 APC and CD69 PE (both from Immunotools) (antibody panel 1), or CD11c APC (Biolegend), CD40 FITC, CD80 FITC and CD86 FITC (all from BD) (antibody panel 2).

Techniques: Activation Assay, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining